nonstructural protein 1 of sars-cov-2 is a potent
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Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor redirectinghost protein synthesis machinery toward viral RNA.
Shuai Yuan, Lei Peng, Jonathan J. Park, Yingxia Hu, Swapnil C. Devarkar, MatthewB. Dong, Qi Shen, Shenping Wu, Sidi Chen, Ivan B. Lomakin, Yong Xiong
PII: S1097-2765(20)30741-3
DOI: https://doi.org/10.1016/j.molcel.2020.10.034
Reference: MOLCEL 7696
To appear in: Molecular Cell
Received Date: 11 August 2020
Revised Date: 5 October 2020
Accepted Date: 22 October 2020
Please cite this article as: Yuan, S., Peng, L., Park, J.J., Hu, Y., Devarkar, S.C., Dong, M.B., Shen,Q., Wu, S., Chen, S., Lomakin, I.B., Xiong, Y., Nonstructural protein 1 of SARS-CoV-2 is a potentpathogenicity factor redirecting host protein synthesis machinery toward viral RNA., Molecular Cell(2020), doi: https://doi.org/10.1016/j.molcel.2020.10.034.
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Nonstructural protein 1 of SARS-CoV-2 is a potent pathogenicity factor 1
redirecting host protein synthesis machinery toward viral RNA. 2
3
Shuai Yuan1,6, Lei Peng2,4,6, Jonathan J. Park2,4, Yingxia Hu1, Swapnil C. Devarkar1, 4 Matthew B. Dong2,4, Qi Shen1, Shenping Wu5, Sidi Chen2,4*, Ivan B. Lomakin3* &Yong 5 Xiong1,7* 6 7 1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, 8 USA. 9 2 Department of Genetics, Yale University School of Medicine, New Haven, CT, 06520, USA. 10 3 Department of Dermatology, Yale university school of medicine, New Haven, CT, 06520, USA. 11 4 Systems Biology Institute, Yale University, West Haven, CT, 06516, USA. 12 5 Department of Pharmacology, Yale University, West Haven, CT, 06516, USA. 13 6 These authors contributed equally. 14 7 Lead contact. 15
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*Correspondence: [email protected] (S.C.), [email protected] (I.L.), 18
[email protected] (Y.X.) 19
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Summary 21
The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural 22
protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet 23
unknown mechanisms. We show here that among all viral proteins, Nsp1 has the 24
largest impact on host viability in the cells of human lung origin. Differential expression 25
analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. 26
Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits 27
translation by plugging the mRNA-entry channel of the 40S. We also determined the 28
structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket 29
paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is 30
nonfunctional due to the incorrect position of the mRNA 3’ region. Our results elucidate 31
the mechanism of host translation inhibition by SARS-CoV-2 and advances the 32
understanding of the impacts from a major pathogenicity factor of SARS-CoV-2. 33
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Introduction 35
SARS-CoV-2, which causes the worldwide COVID-19 pandemic affecting millions of 36
people, belongs to the β-coronaviruses (Coronaviridae Study Group of the International 37
Committee on Taxonomy of, 2020). The virus contains a positive-sense and single-38
stranded RNA that is composed of 5’-UTR, two large overlapping open reading frames 39
(ORF1a and ORF1b), structural and accessory protein genes, and 3’-poly-adenylated 40
tail (Lim et al., 2016). Upon entering the host cells, ORF1a and ORF1b are translated 41
and proteolytically processed by virus-encoded proteinases to produce functional 42
nonstructural proteins (Nsps) that play important roles in the viral infection and RNA 43
genome replication (Masters, 2006). Nsp1 is the first viral gene encoded by ORF1a 44
(Figure 1A) and is among the first proteins to be expressed after infection (Ziebuhr, 45
2005). It was shown that human SARS-CoV and group 2 bat coronavirus Nsp1 plays a 46
key role in suppressing the host gene expression (Kamitani et al., 2006; Narayanan et 47
al., 2008; Tohya et al., 2009). SARS-CoV Nsp1 has been shown to inhibit host gene 48
expression using a two-pronged strategy. Nsp1 targets the 40S ribosomal subunit to 49
stall the translation in multiple steps during initiation of translation and also induces an 50
endonucleolytic cleavage of host RNA to accelerate degradation (Kamitani et al., 2009; 51
Lokugamage et al., 2012). Nsp1 therefore has profound inhibitory effects on the host 52
protein production, including suppressing the innate immune system to facilitate the viral 53
replication (Narayanan et al., 2008) and potentially long-term cell viability 54
consequences. Intriguingly, viral mRNA overcomes this inhibition by a yet unknown 55
mechanism, likely mediated by the conserved 5’ UTR region of viral mRNA (Huang et 56
al., 2011; Tanaka et al., 2012). Taken together, Nsp1 acts as an important factor in viral 57
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lifecycle and immune evasion, and may be an important virulence factor causing the 58
myriad of long-term illnesses of COVID-19 patients. It has been proposed as a target for 59
live attenuated vaccine development (Wathelet et al., 2007; Zust et al., 2007). 60
It is common for RNA viruses to target the initiation step of the host protein 61
translation system to allow expression of the viral proteins (Jan et al., 2016). Most 62
cellular mRNAs have a 5’ 7-methylguanosine (m7G) cap structure, which is essential for 63
mRNA recruitment to the 43S preinitiation complex (PIC) through interaction with the 64
translation initiation factor (eIF) eIF4F. 43S PIC is formed by the 40S ribosomal subunit, 65
the ternary complex eIF2-GTP-Met-tRNAiMet, and the multi-subunit initiation factor eIF3. 66
Binding of the 43S PIC to the m7G-cap results in the loading of the mRNA in the mRNA-67
binding channel of the 40S to form the 48S PIC, and scanning of the mRNA from 5’ to 3’ 68
direction under control of eIF1A and eIF1, until the initiation codon AUG is placed in the 69
P site of the 40S. Base pairing of Met-tRNAiMet with AUG results in conformational 70
changes in the 48S PIC for joining the large 60S ribosomal subunit to form the 80S 71
ribosome primed for protein synthesis (Hinnebusch, 2014, 2017b; Hinnebusch et al., 72
2016). With the exception of type IV IRESes, such as the cricket paralysis virus (CrPV) 73
and Taura syndrome virus (TSV) IRESes, which do not require any host’s eIFs, all other 74
viruses may target different eIFs to redirect the host translational machinery on to their 75
own mRNA (Hertz and Thompson, 2011; Lozano and Martinez-Salas, 2015; Walsh and 76
Mohr, 2011). 77
We present here data demonstrating that among all viral proteins, Nsp1 causes 78
the most severe viability reduction in the cells of human lung origin. The introduction of 79
Nsp1 in human cells broadly alter the transcriptomes by repressing major gene clusters 80
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responsible for protein synthesis, mitochondria function, cell cycle and antigen 81
presentation, while inducing a broad range of factors implicated in transcriptional 82
regulation. We further determined the cryo-EM structures of the Nsp1-40S complex with 83
or without the CrPV IRES RNA, which reveal the mechanism by which Nsp1 inhibits 84
protein synthesis and regulates viral protein production. These results significantly 85
advance our understanding of the Nsp1-induced suppression of host gene expression, 86
the potential mechanisms of SARS-CoV-2 translation initiation, and the broad impact of 87
Nsp1 as a comorbidity-inducing factor. 88
89
Results 90
SARS-CoV-2 open reading frame (ORF) screen identifies Nsp1 as a major viral 91
factor that affects cellular viability 92
A recent study has mapped the interactome of viral protein to host cellular components 93
in human HEK293 cells (Gordon et al., 2020), suggesting that these viral proteins might 94
have diverse ways of interacting or interfering with the fundamental cellular machineries 95
of the host cell. We generated a non-viral over-expression vector (pVPSB) for 96
introduction of viral proteins into mammalian cells and testing their effect on cells 97
(Figure 1B). We first confirmed that the positive control GFP can be introduced into 98
virtually all cells at 100% efficiency, using flow cytometry analysis. We cloned 28 viral 99
proteins (27 of the 29 viral proteins and Nsp5 C145A mutation) as open reading frames 100
(ORFs) into this vector and introduce them into human cells by transfection. Intact 101
cDNAs of Nsp3 and Nsp16 had not been available when we performed the screen and 102
thus were not included in the screen, therefore the cellular phenotypes of these two viral 103
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proteins have not been tested here. We chose to first test H1299, an immortalized 104
cancer cell line of human lung origin. Although H1299 cells are not primary lung 105
epithelial cells, they have been utilized as a cellular model to study SARS-CoV, MERS 106
and SARS-CoV-2 (Hoffmann et al., 2020; Wong et al., 2015). 107
We introduced all 28 cloned ORFs individually in parallel to conduct a mini-108
screen of viral proteins’ effect on the viability of H1299 cells (Figures 1B and 1C). We 109
measured cell viability in two time points, 48 and 72 hours (h) post transfection. 110
Unexpectedly, we found Nsp1 as the sole “hit” with significant effect on cell viability at 111
both time points (Figure 1C). To validate the viability observations with increased 112
sensitivity, we generated an H1299 cell line with a constitutive firefly luciferase reporter 113
(H1299-PL), and confirmed that GFP can also be introduced into this cell line at near 114
100% efficiency (Figures S1A-C). We performed validation experiments, again with all 115
28 ORFs along with vector control, at 3 different time points (24, 48 and 72h). Across all 116
three time points, Nsp1-transfected H1299 cells have dramatically reduced luciferase 117
signal, an approximation of cell numbers (Figure 1D). We further repeat the same 118
experiments with the Vero E6 cell line, an African monkey (Cercopithecus Aethiops) 119
kidney derived cell line, commonly used in SARS-CoV-2 cellular studies (Blanco-Melo 120
et al., 2020; Hoffmann et al., 2020; Kim et al., 2020; Zhou et al., 2020). Consistently, we 121
observed a robust reduction of cellular viability in Vero E6 cells transfected with Nsp1 122
across all 3 time points (Figure S1D). These data revealed that among all SARS-CoV-2 123
proteins, Nsp1 has the largest detrimental effect on cell viability in H1299 and Vero E6 124
cells. 125
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Nsp1 mutants abolish cellular viability phenotype 127
To ensure that the observed reduction of cell viability is indeed from expression of 128
functional Nsp1, we tested three different mutants of Nsp1, including a truncation 129
mutation after residues 12 (N terminal mutant, N-trunc) and two double mutations that 130
have been reported to ablate the activity of SARS-CoV Nsp1 (Wathelet et al., 2007). As 131
SARS-CoV Nsp1 is highly homologous to SARS-CoV-2 Nsp1, we hypothesize that 132
these evolutionarily conserved amino acids may also have significant influence on the 133
activity of SARS-CoV-2 Nsp1. The point mutations include Nsp1 mutant3 that has 134
R124/K125 replaced with S124/E125 (R124S/K125E) and Nsp1 mutant4 that has N128/ 135
K129 replaced with S128/E129 (N128S/K129E). We performed cellular viability assays 136
with wild-type (WT) Nsp1 along with all three of its mutants. In both H1299-PL and Vero 137
E6-PL cells, we again observed that introduction of Nsp1 into cells significantly reduced 138
cell viability along 24, 48, and 72 hours post electroporation (Figures 1E and S1E). 139
Each of the three mutants (truncation, R124S/K125E and N128S/K129E) reverted this 140
phenotype to the vector control level, fully abolishing the cytotoxic effect of Nsp1 141
(Figures 1E and S1E). These results confirmed that functional Nsp1, but not its loss-of-142
function mutants, induce reduction of cellular viability when overexpressed in the two 143
mammalian cell lines. 144
We further tested if Nsp1 expression also leads to cell death. We introduced 145
Nsp1 into H1299 cells, along with controls of empty vector and several other viral 146
proteins (Nsp2, Nsp12, Nsp13, Nsp14, ORF9b, and Spike), and measured cellular 147
apoptosis at 48h post electroporation by flow cytometry analysis of cleaved Caspase 3 148
staining. We found that introduction of Nsp1, but not other viral proteins, induced 149
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apoptosis in H1299 cells (Figure S1G). To ensure the cellular apoptosis effect is indeed 150
from expression of functional Nsp1 protein, we performed the same apoptosis assay 151
with Nsp1 and the three non-functional mutants described above. Consistently, only 152
wild-type (WT) Nsp1 induced apoptosis in H1299-PL cells, whereas the three mutants 153
did not (Figure S1F). Replicates of this cleaved Caspase 3 flow assay with the 154
truncation mutation of Nsp1 confirmed that WT Nsp1, but not the loss-of-function 155
truncation mutant, induced apoptosis in H1299-PL cells (Figures 1F and 1G). 156
157
Transcriptome profiling of Nsp1-overexpressed cells 158
To unbiasedly investigate the global gene expression changes induced by Nsp1 or its 159
loss-of-function mutant form, we performed transcriptome profiling. We first confirmed 160
that Nsp1 is indeed over-expressed in host cells by qPCR using a custom-designed 161
NSP1-specific probe, at both 24 and 48 hours post electroporation (Figure 2A). We then 162
electroporated in quadruplicates for each of Nsp1, its truncation mutant, or vector 163
control plasmid into H1299-PL cells, and collected samples 24 hours post 164
electroporation for mRNA-seq. We collected 24h instead of 48h or 72h samples in order 165
to capture the earlier effect of Nsp1 on cellular transcriptome. We mapped the mRNA-166
seq reads to the human transcriptome and quantified the expression levels of annotated 167
human transcripts and genes (Table S3). Principle component analysis showed clear 168
grouping and separation of WT Nsp1, mutant Nsp1, or vector control groups (Figure 169
2B), confirming the overall quality of the Nsp1 mRNA-seq dataset. 170
Differential expression analysis revealed broad and potent gene expression 171
program changes induced by Nsp1 (Figure 2C; Table S3 and S4), with 5,394 genes 172
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significantly downregulated and 3,868 genes significantly upregulated (FDR adjusted q 173
value < 0.01). To examine the highly differentially expressed genes, we used a highly 174
stringent criteria (FDR adjusted q value < 1e-30), and identified 1,245 highly significantly 175
downregulated genes (top NSP1 repressed genes) and 464 highly significantly 176
upregulated genes (top Nsp1 induced genes) (Figure 2C; Table S3 and S4). In sharp 177
contrast, Nsp1 truncation mutant and the vector control showed no differential 178
expression in the transcriptome, even when using the least stringent criteria (FDR 179
adjusted q value < 0.05) (Figures S2A-B; Table S3 and S4). These data revealed that 180
Nsp1 alone can cause major alterations broadly in the transcriptome shortly (24h) after 181
its introduction into host cells, consistent with its cell viability phenotype (Figure 1). 182
183
Enriched pathway analysis on differentially expressed gene sets revealed strong 184
signatures of cellular transcriptome alterations by Nsp1 185
We globally examined the highly differentially expressed genes as a result of Nsp1 186
expression. To understand what these genes represent as a group, we performed 187
DAVID clustering and biological processes (BP) analysis on the 1,245 top Nsp1-188
repressed genes and the 464 top Nsp1-induced genes, respectively (Figure 2D; Table 189
S4). Enriched pathways in the top Nsp1-repressed genes showed that the most 190
significant gene ontology groups include functional annotation clusters of ribosomal 191
proteins and translation related processes, such as terms of ribonucleoprotein (RNP) 192
(Hypergeometric test, FDR-adjusted q = 6.30e-57), ribosomal RNA processing (q = 193
2.03e-28), and translation (q = 3.93e-28). Highly enriched Nsp1-repressed genes also 194
include the clusters of mitochondria function and metabolism (most terms with q < 1e-195
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15) and cell cycle and cell division (most terms with q < 1e-10), consistent with the 196
reduced cell viability phenotype. Other intriguing enriched Nsp1-repressed pathways 197
include ubiquitin/proteasome pathways and antigen-presentation activities, as well as 198
mRNA processing. We further performed gene set enrichment analysis (GSEA) that 199
takes into consideration both gene set and ranks of enrichment, and the results largely 200
validated the DAVID findings, with highly similar strongly enriched pathways (Figures 3A 201
and S2C). Analysis of highly differentially expressed genes between Nsp1 vs. Nsp1 202
mutant showed results virtually identical to those of Nsp1 vs. vector (Figures S2A-B, 203
Table S4). 204
We then examined the expression levels of the highly differentially expressed 205
genes in the context of enriched pathways in Nsp1, mutant Nsp1, or vector control 206
plasmid in H1299-PL cells. As shown in the heatmaps (Figure 3B), over 70 genes 207
involved in translation are strongly repressed upon introduction of Nsp1, including the 208
RPS, RPL, MRPS, MRPL family members, along with other translational regulators 209
such as AKT1. The repression effect on these genes is completely absent in the Nsp1 210
mutant group (Figure 3B). The strong repression effect also hit multiple members of the 211
gene families involved in mitochondria function, such as the COX, NUDFA, NUDFB and 212
NUDFS families (Figure 3C). Consistent with the cellular phenotypes, Nsp1 also 213
repressed a large number of mitotic cell cycle genes, including members in the CDK, 214
CDC and CCNB families, components of the centrosome, the anaphase promoting 215
complex and various kinases (Figure 3D). While part of the signal may be driven by 216
ribosomal and/or proteosomal genes, multiple genes involved in the mRNA processing 217
and/or nonsense-mediated decay nevertheless are significantly repressed by Nsp1 218
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(Figures S2D-E). Interestingly, DAVID BP enrichment analysis of Nsp1-repressed genes 219
also scored the antigen presentation pathway, mostly proteasome components along 220
with several MHC-I component members (Figure 3E). Concordantly, Nsp1-repressed 221
genes are also enriched in the ubiquitination and proteasome degradation pathways 222
(Figure S2F). 223
On the other hand, genes highly induced by Nsp1 hit a broad range of factors 224
implicated in transcriptional regulation, such as unfolded protein response regulators 225
(ATF4, XBP1), FOX family transcription factors (TFs) (FOXK2, FOXE1, FOXO1, 226
FOXO3), Zinc finger protein genes (ZFN217, ZFN567), KLF family members (KLF2, 227
KLF10), SOX family members (SOX2, SOX4), Homeobox genes (HOXD9, HOXC8, 228
HOXD13), GATA TFs (GATAD2B, GATA6), dead-box protein genes (DDX5, DHX36), 229
cell fate regulators (RUNX2, CREBRF, LIF, JUNB, ELK1, JAG1, SMAD7, BCL3, 230
EOMES); along with certain epigenetic regulators of gene expression such as the 231
SWI/SNF family members ARID1A, ARID1B, ARID3B, and ARID5B (Figure 3F). 232
Interestingly, highly upregulated genes are also slightly enriched in the MAPK/ERK 233
pathway, where Nsp1 expression induces multiple DUSP family members (Figure 3G). 234
The upregulated genes also include several KLF family members related to the process 235
of cellular response to peptide (Figure S2G). Again, the induction effect on these genes 236
is completely abolished in the Nsp1 mutant group (Figures 3F and 3G). These data 237
together showed that Nsp1 expression broadly and significantly altered multiple gene 238
expression programs in the host H1299-PL cells. 239
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Cryo-EM structure reveals Nsp1 is poised to block host mRNA translation. 241
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To elucidate the mechanism of translation inhibition by Nsp1, we determined the cryo-242
EM structure of rabbit 40S ribosomal subunit complex with Nsp1 at 2.7 Å resolution 243
(Table 1, Figure S3). The density observed in the mRNA entry channel enabled us to 244
build an atomic model for the C-terminal domain of Nsp1 (C-Nsp1, amino acids (aa) 245
145-180) (Figure 4A). C-Nsp1 comprises two α-helices (α1, aa 154-160; α2, aa 166-246
179) and two short loops (aa 145-153 and 161-165), which blocks the mRNA entry 247
channel (Figure 4A-B). Besides the α-helices in the mRNA channel, extra globular 248
density between the ribosomal protein uS3 and rRNA helix h16 is observed at a lower 249
contour level, whose dimensions roughly matched the N-terminal domain of Nsp1 (aa: 250
13-127, N-Nsp1, PDB:2HSX) (Almeida et al., 2007) (Figure 4C). However, N-Nsp1 does 251
not appear to be stably bound to the 40S and the low local resolution of the cryo-EM 252
map in this region did not allow for an atomic model for the N-Nsp1. 253
C-Nsp1 bridges the head and body domains of the 40S ribosomal subunit 254
through extensive electrostatic and hydrophobic interactions with the ribosomal proteins 255
uS3 of the head, uS5 and eS30 and helix h18 of the 18S rRNA in the body (Figure 4D). 256
The negatively charged residues D152, E155 and E159 of C-Nsp1 interact with the 257
positively charged residues R117, R116, R143 and K148 of uS3, respectively (Figure 258
4E). In addition, K164 and H165 of Nsp1 inserts into the negatively charged pocket 259
formed by the backbone of U607, G625 and U630 of the rRNA h18. R171 and R175 of 260
C-Nsp1 interact with the negatively charged patch formed by G601, A604, G606 and 261
U607 of h18 (Figure 4E). Besides electrostatic contacts, a large hydrophobic patch of C-262
Nsp1, which is formed by F157, W161, L173 and L177, interacts with a complimentary 263
hydrophobic patch on uS5 formed by V106, I109, P111, T122, F124, V147 and I151 264
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(Figure 4E). Intriguingly, K164 and H165 of Nsp1, which have been shown to play an 265
important role in host translation inhibition, are conserved only in the 266
betacoronaviruses (beta-CoVs) (Figure S4). In addition, the other Nsp1 residues 267
interacting with the h18 of rRNA are also conserved only among the beta-CoVs (Figure 268
S4). This sequence conservation indicates that the hydrophobic interactions between C-269
Nsp1 and uS5 are likely universal in both alpha- and beta-CoVs, while the electrostatic 270
interactions between C-Nsp1 and the h18 of the 18S rRNA are conserved only in the 271
beta-CoVs. The extensive interactions result in C-Nsp1 plugging the mRNA entry 272
channel, which prevents the loading and accommodation of the mRNA (Figure 4B), 273
providing a structural basis for the inhibition of host protein synthesis by Nsp1 of SARS-274
CoV-2 and SARS-CoV reported previously (Kamitani et al., 2009; Kamitani et al., 2006). 275
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Nsp1 locks the 40S in a conformation incompatible with mRNA loading and 277
disrupts initiation factor binding 278
The ribosomal protein uS3 is conserved in all kingdoms. Together with h16, h18 and 279
h34 of 18S rRNA it constitutes the mRNA-binding channel and the mRNA entry site 280
(Graifer et al., 2014; Hinnebusch, 2017a). It has been shown that uS3 interacts with the 281
mRNA and regulates scanning-independent translation on a specific set of mRNAs 282
(Haimov et al., 2017; Sharifulin et al., 2015). Interestingly, conserved residues R116 and 283
R117 of uS3, which are crucial for stabilizing mRNA in the entry channel and 284
maintaining 48S PIC in the closed conformation, are interacting with D152, E155 of 285
Nsp1 in our structure (Dong et al., 2017; Hinnebusch, 2017a) (Figure 4E). Moreover, the 286
conformation of the 40S ribosomal subunit in Nsp1-40S complex is similar to that of 287
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‘closed state’ of 48S PIC with initiator tRNA locked in the P site and the latch closed 288
(Lomakin and Steitz, 2013), which is incapable of mRNA loading. The distance between 289
G610 (h18) and GLN179 (CA, uS3) is shortened from 19.4 Å in the ‘open state’ 48S PIC 290
(PDB:3JAQ) to 15.8 Å in Nsp1-40S ribosomal complex, which is similar to the distance 291
of 15.0 Å in the closed state 48S PIC (PDB:4KZZ) (Figure 4F). This shows that Nsp1 292
not only plugs the mRNA entry channel, but also keeps the 40S subunit in a 293
conformation that is incompatible with mRNA loading. 294
The known structure of the N-terminal domain of SARS-CoV (N-Nsp1) (Almeida 295
et al., 2007) (PDB ID: 2HSX) can be docked into the extra globular density between uS3 296
and rRNA helix h16 in the cryo-EM map (Figure 4G). This potential interaction between 297
N-Nsp1 and uS3 covers most of the uS3 surface on the solvent side, including the 298
GEKG loop of uS3 (aa: 60-63) that corresponds to the consensus GXXG loop 299
conserved in the KH domains of various RNA-binding proteins (Babaylova et al., 2019; 300
Graifer et al., 2014). Mutation of the GEKG loop to alanines does not abrogate the 301
ability of the 40S to bind mRNA and form 48S preinitiation complex (PIC). Instead, it 302
results in the formation of aberrant 48S PIC that cannot join the 60S ribosomal subunit 303
and assemble the 80S initiation complex (Graifer et al., 2014). Peculiarly, binding of 304
SARS-CoV Nsp1 to the ribosome led to the same effect (Kamitani et al., 2009). We 305
hypothesize that Nsp1 may prevent the formation of physiological conformation of the 306
48S PIC induced by uS3 interaction with translation initiation factors, such as the j 307
subunit (eIF3j) of the multi-subunit initiation factor eIF3 (Babaylova et al., 2019; Cate, 308
2017; Sharifulin et al., 2016). The eIF3 complex plays a central role in the formation of 309
the translation initiation complex (Cate, 2017; Hinnebusch, 2014). eIF3j alone binds to 310
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the 40S ribosomal subunit and stabilizes the interaction with eIF3 complex (Fraser et 311
al., 2004; Sokabe and Fraser, 2014). The binding site of eIF3j to 40S subunit is not 312
precisely determined. Cryo-EM and biochemical studies mapped it onto the mRNA 313
binding channel of the 40S, extending from the decoding center toward the mRNA entry 314
region, including the GEKG loop of uS3 (Aylett et al., 2015; Fraser et al., 2007; Hershey, 315
2015) (Figure 4G). 316
We tested if Nsp1 can compete with eIF3j for the binding to the 40S ribosomal 317
subunit. The result showed that Nsp1 indeed significantly reduces the binding between 318
eIF3j and the 40S (Figure 4H). The binding competition of eIF3j and Nsp1 to the 40S 319
was tested at different concentrations. There is little eIF3j binding to the 40S when the 320
concentration of eIF3j is equal or lower than that of Nsp1, and residual eIF3j binding 321
was observed only when its concentration is higher than that of Nsp1 (Figures 4H and 322
S5). By contrast, the binding of Nsp1 to the 40S is not affected even when eIF3j is in 323
excess. These results indicate that Nsp1 disrupts the binding of eIF3j to the 40S, 324
potentially by shielding the access to uS3 and the mRNA binding channel and/or by 325
making the conformation of the 40S unfavorable for eIF3j interaction. 326
327
Nsp1 prevents physiological conformation of the 48S PIC 328
It was shown previously that binding of SARS-CoV Nsp1 to the 40S ribosomal subunit 329
does not inhibit 48S PIC formation, but it suppresses 60S subunit joining (Kamitani et 330
al., 2009). To understand the effect of Nsp1 of SARS-CoV-2 on 48S PIC, we determined 331
a 3.3 Å resolution cryo-EM structure of Nsp1 bound to the 48S PIC assembled with the 332
cricket paralysis virus (CrPV) internal ribosome entry site (IRES) (Figures 5A and S6). 333
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CrPV IRES has become an important model for studies of the eukaryotic ribosome 334
during initiation, as it is able to directly recruit and assemble with 40S or 80S ribosome 335
without requiring any eIFs (Martinez-Salas et al., 2018). It was shown that SARS-CoV 336
Nsp1 inhibits translation of the CrPV IRES RNA (Kamitani et al., 2009). The use of 337
CrPV IRES allowed us to probe if Nsp1 completely inhibits mRNA binding to the 40S 338
subunit or it acts on the mRNA entry site only, as binding of the IRES may help fix 5’-339
region of the mRNA on the ribosome mRNA exit region, enabling the investigation of the 340
mRNA path on the 40S subunit in the presence of Nsp1. We first examined whether 341
Nsp1 affects binding of the IRES RNA to the 40S ribosomal subunit. The result shows 342
that Nsp1 and CrPV IRES can bind 40S ribosomal subunit simultaneously (Figure S6A-343
B). Consistently, both C-Nsp1 and the CrPV IRES can be seen in the cryo-EM map 344
(Figure 5A), where the Nsp1 C-terminal domain is inserted in the RNA entry channel in 345
the same way as in the Nsp1-40S complex without the IRES RNA (Figures 4A and 4B). 346
The local environment of C-Nsp1 in the ribosome RNA entry channel with or without the 347
IRES RNA is quite similar. No conformational changes were observed for C-Nsp1, 348
protein uS5 and rRNA h18, however, the head of the 40S subunit is moved by about 2.8 349
Å (Figure 5A) (discussed more below). 350
We fitted the high resolution structure of the CrPV IRES from the yeast 40S-CrPV 351
IRES complex(Murray et al., 2016) (PDB: 5IT9) into our cryo-EM map. Importantly, the 352
pseudoknot I (PKI) domain of the CrPV IRES, which is a structural mimic of the 353
canonical tRNA-mRNA interaction, is not seen in the cryo-EM map, suggesting that it is 354
dislodged from the 40S in the presence of Nsp1 (Figure 5B). Consistently, there would 355
be a clash between Nsp1 C-terminal domain and the 3’ region of the IRES RNA in the 356
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previously observed conformation bound to the 40S (Murray et al., 2016) (Figure 5B). 357
The conformation of the 40S head in the Nsp1-40S-CrPV IRES complex is different 358
from that in the Nsp1-40S complex (Figure 5C). The head in the Nsp1-40S-CrPV IRES 359
complex is in somewhat intermediate conformation compared to the Nsp1-40S and the 360
40S-CrPV IRES complexes (Figure 5C). This suggests that the Nsp1-40S interactions 361
resist the conformational changes induced by the IRES for translation initiation. 362
Conformational changes of the head domain of the 40S subunit play important role in 363
the mRNA loading and recruitment of the 60S subunit to form the 80S ribosome. Nsp1 364
limits the rotation of the head, which may have profound consequences interfering with 365
the joining of the 60S subunit and the formation of the 80S initiation complex. 366
367
Discussion 368
Viral infection is a complex process involving multiple components and certain viral 369
proteins are often in high abundance in cells during active viral replication (Astuti and 370
Ysrafil, 2020; Yoshimoto, 2020). Therefore, understanding the effects of each individual 371
viral protein on the cells provides important insights on the cellular impacts of viral 372
infection. Using a reductionist approach, we tested the gross cellular effect of 373
expressing most of the SARS-CoV-2 proteins individually, and found that among all 374
ORFs tested, Nsp1 showed the strongest deleterious effect on cell viability in H1299 375
cells of human lung epithelial origin. This is in concordance with previous observations 376
from related coronaviruses, such as mouse hepatitis virus (MHV) Nsp1 being a major 377
pathogenicity factor strongly reducing cellular gene expression (Zust et al., 2007), and 378
SARS-CoV Nsp1 inhibiting interferon (IFN)-dependent signaling and having significant 379
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effects on cell cycle (Wathelet et al., 2007). A recent study shows that SARS-CoV-2 380
Nsp1 shuts down mRNA translation in cells and suppresses innate immunity genes 381
such as IFNb and IL-8, although these experiments were conducted in HEK293T cells 382
of kidney origin, and only a small number of host genes were tested (Thoms et al., 383
2020b). As an unbiased interrogation of global cellular pathways affected by Nsp1, our 384
transcriptome profiling data and gene set enrichment analysis revealed strong 385
signatures of transcriptomic changes in broad ranges of host genes with several major 386
clusters, providing a comprehensive understanding of the impacts of one of the most 387
potent pathogenicity protein factors of SARS-CoV-2 in human cells of lung origin. 388
Our structure of the SARS-CoV-2 Nsp1 protein bound to the 40S ribosomal 389
subunit establishes a mechanistic basis of the cellular effects of Nsp1, revealing a 390
multifaceted mechanism of inhibition of the host protein synthesis at the initiation stage 391
by the virus. Nsp1 plugs the mRNA channel entry, which physically blocks access to the 392
channel by any mRNA (Figure 4B). Moreover, Nsp1 locks the head domain of the 40S 393
subunit in the closed position, characterized by the closed conformation of the “mRNA 394
entry channel latch” that clams around incoming mRNA (Hinnebusch, 2017b; Lomakin 395
and Steitz, 2013; Passmore et al., 2007). The latch is supposed to be closed during the 396
scanning of the mRNA, keeping mRNA locked in the binding cleft and increasing 397
processivity of the scanning, whereas the open conformation of the latch would facilitate 398
the initial attachment of the 43S PIC to the mRNA (Lomakin and Steitz, 2013). 399
Therefore, when Nsp1 keeps the latch closed it makes impossible for the host mRNA to 400
be loaded. In addition, we showed that Nsp1 competes with eIF3j for the binding to the 401
40S subunit (Figure 4H). This allows us to propose that Nsp1 weakens the binding of 402
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the eIF3 to the 40S subunit by disrupting uS3-eIF3j interaction. Recently, several 403
structures of Nsp1 bound ribosomal complexes were reported, including binary (Nsp1-404
40S), with ribosome biogenesis factor TSR1, and with eIF3-containing PICs (Schubert 405
et al., 2020; Thoms et al., 2020a). None of these structures, however, captured the 406
mRNA, which likely is flexible or dissociates from the PIC because of the lack of the 407
mRNA-eIF4F interaction. Using CrPV IRES RNA we were able to visualize the RNA 408
bound to Nsp1-40S complex and show that Nsp1 does not inhibit mRNA binding to the 409
ribosome, instead it prevents physiological conformation of the 48S PIC by restricting 410
the ribosome head domain rotation. 411
Our results explain how Nsp1 inhibits protein synthesis; however, how SARS-412
CoV-2 escapes this inhibition and initiate translation of its own RNA still remains 413
unanswered. The 5’-UTR of SARS-CoV is essential for escaping Nsp1-mediated 414
suppression of translation (Tanaka et al., 2012). Interactions involving the viral 5’ UTR 415
presumably result in the “unplugging” of Nsp1 from the 40S ribosome during the 416
initiation of viral translation. In addition, the weakening of eIF3 binding to the 40S 417
subunit is beneficial for translation initiation of some viruses. The hepatitis C virus (HCV) 418
IRES displaces eIF3 from the interface of the 40S subunit to load its RNA in the mRNA 419
binding channel (Hashem et al., 2013; Niepmann and Gerresheim, 2020). HCV IRES 420
interacts with eIF3a, eIF3c and other core subunits of eIF3 to promote formation of the 421
viral 48S PIC (Cate, 2017). The eIF3d subunit of the eIF3 complex can be cross-linked 422
to the mRNA in the exit channel of the 48S PIC, it has its own cap-binding activity which 423
can replace canonical eIF4E dependent pathway and promote translation of selected 424
cellular mRNAs (Lee et al., 2016; Pisarev et al., 2008; Walker et al., 2020). Interestingly, 425
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a recent genome-wide CRISPR screen revealed the eIF3a and eIF3d are essential for 426
SARS-CoV-2 infection (Wei et al., 2020). The requirement of the same essential 427
initiation factors suggests that it is possible that SARS-CoV-2 may use an “IRES-like” 428
mechanism involving eIF3 recruitment by 5’ UTR to overcome Nsp1 inhibition. Binding 429
of 5’ UTR may cause conformational change of the 40S head leading to the latch 430
opening, Nsp1 dissociation, viral RNA loading into mRNA binding channel and formation 431
of the functional 80S initiation complex primed for viral protein synthesis. However, the 432
detailed mechanisms of viral escape of Nsp1 inhibition must await for future 433
experimental studies. 434
435
Limitations 436
The transcriptome changes were observed in the presence of Nsp1 in the cells of 437
human lung origin. However, the role of the transcriptome changes in the loss of cell 438
viability is still not understood. To elucidate the mechanism of translation inhibition by 439
Nsp1, we determined the cryo-EM structure of rabbit 40S ribosomal subunit complex 440
with Nsp1. The atomic structure of C-Nsp1 was built into well-defined high-resolution 441
density, while only global density of the N-Nsp1 was observed. Further work is needed 442
to reveal the details and the potential functional consequence of the interaction of N-443
Nsp1 and 40S ribosome subunit. Our results suggested potential mechanisms of SARS-444
CoV-2 translation initiation, but future experiments are needed to illustrate how SARS-445
CoV-2 overcomes the Nsp1 inhibition and starts the translation of its own genome. 446
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Acknowledgements 447
We thank the Yale cryo-EM facilities for assistance with data collection. We thank the 448
Xiong lab and Chen lab members for discussions. We thank Drs. Krogan and Gordon 449
for generously providing the initial cDNA templates of SARS-CoV-2 ORFs. We thank 450
Xiaoyun Dai, Lupeng Ye, Paul Clark and several others for sharing various plasmids 451
and reagents. We thank the Yale Center for Genome Analysis and Center for Research 452
Computing for providing High-throughput sequencing and computing assistance and 453
resources. This work was supported by Yale discretionary funds to Y.X. and S.C. The 454
lab of SC is also supported by NIH (DP2CA238295, 1R01CA231112, U54CA209992-455
8697, R33CA225498, RF1DA048811) and DoD (W81XWH2010072). 456
457
Author contributions 458
S.Y., L.P., S.C., I.B.L. and Y.X. initiated the project and designed the experiments. S.Y. 459
I.B.L, Q.S. and Y.H. produced proteins and 40S ribosomal subunit. S.Y. and S.D. 460
performed binding assays. S.Y. prepared the cryo-EM samples. Y.H. and S.W. carried 461
out cryo-EM data collection. S.Y. and Y.X. did cryo-EM data processing. S.Y., I.B.L., 462
S.D. and Y.X. analyzed cryo-EM structure. L.P. and M.B.D. performed cellar assays. 463
L.P. and J.J.P. performed and processed mRNA-seq. S.Y., L.P., S.C., I.B.L. and Y.X. 464
prepared the manuscript. S.C., I.B.L. and Y.X. jointly supervised the work. 465
466
Declaration of interests 467
The authors declare no competing interests. 468
469
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Figures 470
471
Figure 1. SARS-CoV-2 ORF mini-screen identified Nsp1 as a key viral protein with 472
host cell viability effect. 473
(A) Schematics of viral protein coding frames along SARS-CoV-2 genome. Colored 474
ORFs indicate the ones used in this study, while two ORFs in grey are not (Nsp3 475
and Nsp16). 476
(B) Schematics of molecular and cellular experiments of viral proteins. 477
(C) Scatter plot of SARS-CoV-2 ORF mini-screen for host viability effect in H1299 478
cells, at 48 and 72 hours post ORF introduction. Each dot represents the mean 479
normalized relative viability of host cells transfected with a viral protein encoding 480
ORF. Dash line error bars indicate standard deviations. (n = 3 replicates). Pink 481
color indicates hits with p < 0.05 (one-way ANOVA, with multiple group 482
comparison). 483
(D) Bar plot of firefly luciferase reporter measurement of viability effects of SARS-484
CoV-2 ORFs in H1299-PL cells, at 24, 48 and 72 hours post ORF introduction (n 485
= 3 replicates). 486
(E) Bar plot of firefly luciferase reporter measurement of viability effects of Nsp1 and 487
three Nsp1 mutants (truncation, mut3: R124S/K125E and mut4: N128S/K129E) 488
in H1299-PL cells, at 24, 48 and 72 hours post ORF introduction (left, middle and 489
right panels, respectively) (n = 3 replicates). 490
(F) Flow cytometry plots of apoptosis analysis of Nsp1 and loss-of-function 491
truncation mutant in H1299-PL cells, at 48 hours post ORF introduction. 492
Percentage of apoptotic cells was gated as cleaved Caspase 3 positive cells. 493
(G) Quantification of flow-based apoptosis analysis of Nsp1 and loss-of-function 494
truncation mutant in H1299-PL cells, at 48 hours post ORF introduction. 495
For all bar plots in this figure: Bar height represents mean value and error bars 496
indicate standard error of the mean (sem). (n = 3 replicates for each group). 497
Statistical significance was accessed by ordinary one-way ANOVA, with multiple 498
group comparisons where each group was compared to empty vector control, with p-499
values subjected to multiple-testing correction by FDR method. (ns, not significant; * 500
p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). 501
See also Figure S1. 502
503
504
Figure 2. Transcriptome profiling of H1299 cells introduced with NSP1 and NSP1 505
truncation mutant by RNA-seq. 506
(A) Quantitative PCR (qPCR) confirmation of NSP1 overexpression, at 24 and 48 507
hours post electroporation. (n = 3 replicates). 508
(B) Principle component analysis (PCA) plot of the entire mRNA-seq dataset, 509
showing separation between Nsp1, Vector control and Nsp1 truncation mutant 510
groups, all electroporated into H1299-PL cells and harvested 24 hours post 511
electroporation. RNA samples were collected as quadruplicates (n = 4 each 512
group). 513
(C) Volcano plot of differential expression between of Nsp1 vs Vector Control 514
electroporated cells. Top differentially expressed genes (FDR adjusted q value < 515
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1e-100) are shown with gene names. Upregulated genes are shown in orange. 516
Downregulated genes are shown in blue. 517
(D) Bar plot of top enriched pathway analysis by DAVID Biological Processes (BP). 518
Nsp1 vs Vector control (top), or Nsp1 vs Nsp1 mutant (bottom), highly 519
downregulated (left) and upregulated (right) genes are shown (q < 1e-30). 520
See also Figure S2 521
522
523
Figure 3. Highly differentially expressed genes between Nsp1, Vector control and 524
Nsp1 mutant group in the context of top major enriched pathways. 525
(A) Gene set enrichment plots of representative enriched pathways by GSEA. 526
(B-E) Heatmap of Nsp1 highly repressed genes (q < 1e-30) in rRNA processing and 527
translation (B), mitochondria function (C), cell cycle (D), MHC-I antigen presentation 528
processes (E). 529
(F-G) Heatmap of Nsp1 highly induced genes (q < 1e-30) in polII related 530
transcription regulation processes (F) and the MAPK/ERK pathway (G). 531
See also Figure S2 532
533
534
Figure 4. cryo-EM structure of the Nsp1-40S ribosome complex. 535
(A) Overall density of the Nsp1-40S ribosome complex with Nsp1 (green) and 40S. 536
ribosome (gray). Inset shows C-Nsp1 with corresponding density with clear 537
sidechain features. C-Nsp1 α-helices (α1, aa 154-160; α2, aa 166-179) are 538
labeled. 539
(B) Cross section of the C-Nsp1 (green) within the mRNA entry channel. 40S. 540
ribosome is shown in surface and C-Nsp1 is displayed in cartoon. 541
(C) Overall density of Nsp1-40S ribosome complex at a lower contour level. Insets. 542
shows the extra globular density with SARS-CoV Nsp1 N-terminal domain 543
(PDB:2HSX, green) fitted. Ribosomal protein uS3 (magenta) and rRNA h16 544
(orange) are shown in cartoon. 545
(D) Overall structure of the C-Nsp1-40S ribosome complex, with C-Nsp1 (green. 546
surface) and the surrounding protein uS3 (magenta sphere representation), uS5 547
(cyan) and rRNA h18 (orange) highlighted. The inset shows zoomed-in view of 548
C-Nsp1 in cartoon, with the surrounding 40S components in cartoon and surface 549
to illustrate the mRNA entry channel. 550
(E) Molecular interactions between C-Nsp1 and 40S ribosome components, 551
including uS3, h18, uS5. Proteins and rRNA are in the same color as in (D) and 552
shown in cartoon, with binding pocket and hydrophobic interface depicted in 553
surface. The interacting residues are shown in sticks. 554
(F) The conformation of the 40S ribosome in the Nsp1-40S complex is similar to the. 555
close form in the 48S PIC. Q179 of uS3 (magenta cartoon) is displayed as a 556
sphere. h18 is in cartoon and colored dark yellow (48S closed conformation), 557
orange (Nsp1-40S ribosome complex) and dark green (48S open conformation), 558
with distances to Q179 indicated by the dashes. 559
(G) The N-terminal domain of Nsp1 covers uS3 surface on the solvent side. The 560
cryo-EM density in this region is shown in blue surface with SARS-CoV Nsp1 561
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N-terminal domain (PDB:2HSX) fitted. uS3 (magenta) is depicted cartoon. The 562
GEKG loop (dark purple) is shown in sphere representation. The putative 563
location of eIF3j is marked in red. 564
(H) SDS-PAGE analysis of Nsp1 and eIF3j competition at different concentration 565
ratios (indicated in the top table). 566
See also Figures S3, S4 and S5. 567
568
569
Figure 5. Nsp1 prevents physiological conformation of the 48S PIC. 570
(A) Overall structure of the Nsp1-40S-CrPV IRES complex. Nsp1 (green) and 571
IRES. (yellow) are presented in surface. The ribosome proteins (slate) and 572
rRNA (orange) are shown in cartoon. The right insets display the conformation 573
change in the Nsp1-binding region (cartoon representation) with or without the 574
IRES. 575
(B) The previously reported model of CrPV IRES (PDB: 5IT9, orange cartoon) fitted 576
to 40S ribosome in the present of Nsp1 (green cartoon). 40S ribosome (slate) 577
and the currently observed IRES (yellow) are presented in surface. 578
(C) C-Nsp1 restricts the 40S ribosome head rotation. Superposition of the Nsp1-579
40S, Nsp1-40S-CrPV IRES and IRES-40S (PDB:5IT9) complexes is shown is 580
cartoon. Zoomed view displays the head rotations represented by selected 581
rRNA regions. C-Nsp1 (green) is displayed in surface. 582
See also Figure S6. 583
584
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Table 1. Cryo-EM data collection, refinement and validation statistics, see also 585
Figure S3 and S6. 586
587
Nsp1-40S ribosome (EMDB-22432) (PDB 7JQB)
Nsp1-40S-CrPV IRES (EMDB-22433) (PDB 7JQC)
Data collection and processing Magnification 81,000 81,000 Voltage (kV) 300 300 Electron exposure (e–/Å2) 50 50 Defocus range (μm) 0.5-2.0 0.5-2.0 Pixel size (Å) 1.068 1.068 Symmetry imposed C1 C1 Initial particle images (no.) 668,695 60,690 Final particle images (no.) 353,927 48,689 Map resolution (Å) 2.7 3.3
FSC threshold. 0.143 0.143 Map resolution range (Å) 2.5-4.5 3.0-5.0 Refinement Initial model used (PDB code) 4KZX 4KZX Model resolution (Å) FSC threshold
2.7 0.143
3.3 0.143
Model resolution range (Å) Map sharpening B factor (Å2) 88 23 Model composition Non-hydrogen atoms Protein residues Ligands (nucleotide)
74,976 4,859 1,697
77,833 4,837 1,840
B factors (Å2) Protein Ligand (nucleotide)
140 150
140 167
R.m.s. deviations Bond lengths (Å) Bond angles (°)
0.007 0.8
0.006 0.9
Validation MolProbity score Clashscore Poor rotamers (%)
1.8 6.4 0.4
1.9 7.9 0.5
Ramachandran plot Favored (%) Allowed (%) Disallowed (%)
93.03 6.91 0.06
92.28 7.55 0.17
588 589
590
591
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STAR Methods. 592
593
RESOURCE AVAILABILITY 594
Lead Contact 595
Further information and requests for resources and reagents should be directed to and 596
will be fulfilled by the Lead Contact, Yong Xiong ([email protected]). 597
Material Availability 598
All unique/stable reagents generated in this study are available from the Lead Contact. 599
Data and Code Availability 600
All data generated or analyzed during this study are included in this article and its 601
supplementary information files. Specifically, source data and statistics for non-high-602
throughput experiments are provided in a supplementary table excel file (Table S2). 603
High-throughput experiment data are provided as processed quantifications in 604
Supplemental Datasets (Table S3 and S4). Genomic sequencing raw data are 605
deposited to NIH Sequence Read Archive (SRA) and/or Gene Expression Omnibus 606
(GEO) and the accession code is PRJNA667046. Constructs are available at either 607
through a public repository or via requests to the corresponding authors. Original cell 608
lines are available at commercial sources listed in supplementary information files. 609
Genetically modified cell lines are available via the authors’ laboratories. Codes that 610
support the findings of this research are being deposited to a public repository such as 611
GitHub, and are available from the corresponding authors upon reasonable request. 612
The cryo-EM maps of the Nsp1-40S ribosome complex and the Nsp1-40S-CrPV 613
IRES ribosome complex have been deposited in the Electron Microscopy Data Bank as 614
EMD-22432 and EMD-22433, respectively. The corresponding structure models are in 615
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the Protein Data Bank with accession code 7JQB, 7JQC. Additional Supplemental Items 616
are available from Mendeley Data at http://dx.doi.org/10.17632/642gjvx74d.1. 617
618
EXPERIMENTAL MODEL AND SUBJECT DETAILS 619
Mammalian cells 620
H1299, H1299-PL, Vero E6, Vero E6-PL cell lines were used in the cell viability assay 621
and the mRNA sequencing. 622
E. coli 623
E. coli BL21(DE3) was used for the expression of recombinant Nsp1 and eIF3j. 624
625
METHOD DETAILS 626
SARS-CoV-2 plasmid cloning 627
The initial cDNA templates of SARS-CoV-2 ORF gene containing plasmids were 628
provided by Dr. Krogan as a gift (Gordon et al., 2020), where the ORFs were primarily 629
cloned into lentiviral expression vector. A non-viral expression vector, pVPSB empty, 630
where ORFs were driven by a constitutive EFS promoter and terminated by a short poly 631
A, was constructed by cloning gBlock fragments (IDT) into pcDNA3.1 vector (Addgene, 632
#52535) by the Gibson assembly (NEB). All ORFs gene encoding fragments were PCR 633
amplified from the lentiviral vectors with ORF-specific forward primers and common 634
reverse primer that containing overlaps that corresponded to flanking sequences of the 635
and KpnI and XhoI restriction sites in the pVPSB empty vector. The primer lists were 636
provided in Table S1. ORFs PCR amplified fragments were gel-purified and cloned into 637
restriction enzyme digested backbone by the Gibson assembly (NEB). A lentiviral vector 638
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constitutively expressing a Firefly Luciferase and a puromycin mammalian selection 639
marker (Lenti-Fluc-Puro) was generated by standard molecular cloning. All plasmids 640
were sequenced and harvested by Maxiprep for following assay. 641
Nsp1 mutant ORF construction 642
Truncation mutant Nsp1 has triple stop codons introduced after residues 12 (N terminal 643
mutant). Nsp1 mutant3 has R124 and K125 replaced with S124 and E125 644
(R124S/K125E). Nsp1 mutant4 has N128 and K129 were converted to S128 and E129 645
(N128S/K129E). IDT gBlocks were ordered for truncated Nsp1 and different Nsp1 646
mutants with 19~23 bp overlaps that corresponded to flanking sequences of the and 647
AgeI and BstXI restriction sites in the pVPSBA01-Nsp1 plasmid. pVPSBA01-Nsp1 648
plasmid were digested and gel purified, and gBlocks were cloned using the Gibson 649
assembly (NEB). 650
Generation of stable cell lines 651
Lentivirus was produced by transfection of co-transgene plasmid (Lenti-Fluc-Puro) and 652
packaging plasmids (psPAX2, pMD2.G) into HEK293FT cells, followed by supernatant 653
harvesting, filtering and concentration with Amicon filters (Sigma). H1299 and Vero E6 654
cells were infected with Lenti-Fluc-Puro lentivirus. After 24 h of virus transduction, cells 655
were selected with 10 µg/mL puromycin, until all cells died in the control group. Luc 656
expressing H1299 and Vero E6 that with puromycin resistance cell lines were obtained 657
and named as H1299-PL and Vero E6-PL (Vero E6-PL for short) respectively. 658
Mammalian cell culture 659
H1299, H1299-PL, Vero E6, Vero E6-PL cell lines were cultured in Dulbecco’s 660
modified Eagle’s medium (DMEM; Thermo fisher) supplemented with 10% Fetal 661
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bovine serum (FBS, Hyclone),1% penicillin-streptomycin (Gibco), named as D10 662
medium. Cells were typically passaged every 1-2 days at a split ratio of 1:2 or 1:4 when 663
the confluency reached at 80%. 664
SARS-CoV-2 ORF mini-screen for cell viability 665
H1299 cells were plated in white opaque walled microwell assay plates, 25,000 cells per 666
96 well. SARS-CoV-2 ORF plasmids, 1 µg of each, were parallelly transfected with 1 µl 667
lipofectamine 2000, in triplicates. Cell viability was detected at every 24hr after 668
transfection using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega). 669
Relative viability was normalized to the mean viability of empty vector transfected 670
control group. All procedures followed the manufacturer standard protocol. Luminescent 671
signals were measured by a Plate Reader (PerkinElmer). 672
Determination of luciferase reporter cell viability 673
H1299-PL and Vero E6-PL cells were plated in white opaque walled microwell assay 674
plates, 25,000 cells per well in a 96 well. SARS-CoV-2 ORF plasmids, 1 µg of each, 675
were parallelly transfected with 1ul lipofectamine 2000. Cell viability was measured 676
every 24 hr after plasmid transfection by adding 150 µg / ml D-Luciferin (PerkinElmer) 677
using a multi-channel pipette. Luciferase intensity was measured by a Plate Reader 678
(PerkinElmer). 679
Electroporation with 4D nucleofection 680
Cells were trypsinized and collected, 1e6 cells were resuspended in SF cell line 681
NucleofectorTM solution with 3 µg plasmid DNA. Cells were transferred into 100 µl 682
NucleocuvetteTM Vessel and NCI-H1299 [H1299] cell specific protocol were utilized 683
according to the manufacturer’s protocol (4D-NucleofectorTM X Unit, Lonza). After the 684
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pulse application, 100 µl prewarmed D10 medium was added to the electroporated cells 685
in the cuvette. Cells were gently resuspended in the cuvette and transferred into 6 well 686
plate, cultured in incubator. Cells were collected at 24 or 48 hours later for 687
flowcytometry assay and RNA extraction. 688
Apoptosis flow cytometry assay 689
Flow cytometry was performed using standard immunology protocols. Briefly, 690
experimental and control cells were electroporated with respective plasmids. After a 691
defined time point, cells were collected, fixed and permeabilized using 692
Fixation/Permeablization Solution kit (BD). Then antigen-specific antibodies with 693
specific dilutions were added into cells and incubated for 30 min on ice. Cells were 694
washed with cold MACS buffer for 3 times before analyzed on a BD FACSAria 695
cytometer. Antibody used: anti-cleaved Caspase-3(Asp175) (Sigma, 9669s, 1:200). 696
Gene expression analysis by mRNA sequencing (mRNA-seq, RNA-seq) 697
For H1299-PL cells electroporated with Nsp1 or Nsp1 mutant, mRNA-seq libraries were 698
prepared following next-generation sequencing (NGS) protocols. Briefly, 1e6 H1299 699
cells were electroporated with 3 µg Nsp1, mutant Nsp1, and relative control plasmids. 700
Electroporation was done in with quadruplicates for each group. Cells were collected 701
24hr post electroporation. Total mRNA was extracted with RNasy Plus Mini Kit (Qiagen). 702
1µg total mRNA each sample was used for the RNA-seq library preparations. A 703
NEBNext® Ultra™ RNA Library Prep Kit for Illumina was employed to perform RNA-seq 704
library preparation and samples were multiplexed using barcoded primers provided by 705
NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1). All procedures follow 706
the manufacturer standard protocol. Libraries were sequenced with Novaseq system 707
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(Illumina). 708
mRNA-seq data processing, differential expression analysis and pathway 709
analysis 710
The mRNA data processing, transcript quantification, differential expression, and 711
pathway analysis were performed using custom computational programs. In brief, Fastq 712
files from mRNA-seq were used analyzed using the Kallisto quant algorithm for 713
transcript quantification (Bray et al., 2016). Differential expression analysis was 714
performed using Sleuth (Pimentel et al., 2017). Z-scores for time course heatmap were 715
calculated by log2-normalizion of gene counts following by scaling by genes. 716
Visualizations of differentially expressed genes such as volcano plots and heatmaps 717
were generated using standard R packages. Differentially upregulated and 718
downregulated genes were subjected to pathway analysis by DAVID (Huang et al., 719
2007) and/or GSEA (Subramanian et al., 2005). Processed mRNA-seq data, differential 720
expression analysis and pathway analysis results are provided in (Table S3 and S4). 721
RT-qPCR 722
Total RNA was extracted from cells using RNasy Plus Mini Kit (Qiagen). Total mRNA 723
was reverse transcribed into cDNA by M-MLV Reverse Transcriptase (Sigma). Samples 724
were collected in triplicates. Gene expression was quantified using Taqman Fast 725
Universal PCR Master Mix (Thermo Fisher) and Taqman probes (Invitrogen). NSP1 726
probe was generated with custom designed according to the Nsp1 DNA sequence in the 727
SARS-CoV-2 genome annotation (2019-nCoV/USA-WA1/2020, accession MN985325). 728
RNA expression level was normalized to ACTB (human). Relative mRNA expression 729
was determined via the ∆∆ Ct method. 730
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Ribosome and CrPV IRES purification 731
40S ribosomal subunits were purified from the rabbit reticulocyte lysate (Green 732
Hectares, USA) as described previously (Lomakin and Steitz, 2013). The gene for wild-733
type CrPV IRES (nucleotides 6028-6240) was chemically synthesized and cloned in the 734
pBluescript SK vector flanked at the 5’-end by a T7 promoter sequence and an EcoRI 735
cleavage site at the 3’-end. Standard in vitro transcription protocol was used for IRES 736
RNA synthesis and purification (MEGAscript™ T7 Transcription Kit, Ambion, USA). 737
Protein construction, expression and purification 738
Full-length SARS-CoV-2 Nsp1 was cloned into pMAT-9s vector and pET-Duet vector for 739
expression of MBP-tagged and 6×his tagged proteins, respectively. The Escherichia coli 740
BL21 (DE3) cells were used for protein expressions, which were induced by 0.5 mM 741
isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 16 hours in Terrific Broth. 742
Cells were harvested and lysed using a microfluidizer. The lysate was clarified by 743
centrifugation and then applied to a Ni-NTA (Qiagen) column. Anion exchange (HiTrap 744
Q HP, GE healthcare) chromatography was performed in a buffer of 50 mM Tris, pH 8.0 745
with a NaCl concentration gradient from 50 mM to 1M. Subsequent size exclusion 746
chromatography (HiLoad Superdex 75, GE healthcare) was performed in a buffer of 50 747
mM Tris, 150 mM NaCl, pH 8.0. Purity of the proteins was analyzed by SDS-PAGE after 748
each step. Full length eIF3j was expressed in Escherichia coli BL21 and purified with a 749
similar method. 750
Filter binding assays 751
Rabbit 40S ribosome and binding partners (proteins or CrPV IRES RNA) were 752
incubated together for 20 min at 37 °C in a total volume of 20 µl in 1× 48S buffer (20 753
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mM HEPES(KOH) pH 7.5, 100 mM KCl, 2.5 mM MgAc, 1 mM DTT, 250 µM Spermidine 754
3HCl). Reaction mixtures were incubated for another 20 min at room temperature 755
before diluting to 100 µl with H100 buffer (10 mM HEPES(KOH) pH 7.0, 100 mM KCl, 5 756
mM MgAc, 2 mM DTT). Diluted reaction mixtures were filtered through 100 kDa filter 757
(Thermo Scientific) in 10,000g for 5 min. The flow through was collected. 200 µl H100 758
buffer was used for washing the unbound proteins or RNA for 4 times before analyzing 759
by SDS-PAGE or RNA gel. 760
The concentration for the 40S ribosome for the filter binding assay is 1.5 µM and 761
the Nsp1 concentration is 15 µM (ratio of 1:10). In the Nsp1 and eIF3j competition 762
assays, the concentrations of eIF3j are 7.5 µM, 15 µM and 30 µM corresponding to 763
ratios of 1:5, 1:10 and 1:20. The concentration of the CrPV IRES is 7.5 µM in the Nsp1-764
IRES binding assay (ratio of 1:5). 765
Cryo-EM sample preparation, data collection and processing 766
40S ribosome and Nsp1, with or without the CrPV IRES RNA were mixed and incubated 767
at 37 °C for 20 mins to form a stable complex. The complex (4 µl) was applied to a C-768
Flat 2/1 3C copper grid (Electron Microscopy Sciences) pretreated by glow-discharging 769
at 8 mA for 20 seconds. The grid was blotted at 20 °C with 100% humidity and plunge-770
frozen in liquid ethane using FEI Vitrobot Mark IV (Thermo Fisher). The grids were 771
stored in liquid nitrogen before data collection. 772
Images were acquired on a FEI Titan Krios electron microscope (Thermo Fisher) 773
equipped with a post-GIF Gatan K3 direct detector in super-resolution mode, at a 774
nominal calibrated magnification of 81,000× with the physical pixel size corresponding 775
to 1.068Å. Automated data collection was performed using SerialEM (Mastronarde, 776
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34
2005). 777
A total of 4,700 movie series were collected for the Nsp1-40S ribosome complex. 778
300 movies series were collected for the Nsp1-40S-CrPV IRES complex. For the Nsp1-779
40S ribosome complex, a defocus range of 0.5 µm to 2 µm was used. Data were 780
collected with a dose of 15.9 electrons per pixel per second. Images were recorded over 781
a 3.6s exposure with 0.1s for each frame to give a total dose of 50 electrons per Å2. 782
Similar conditions were used for the Nsp1-40S-CrPV IRES complex. 783
The same data processing procedures were carried out for both the two 784
complexes using standard pipelines in cryoSPARC(Punjani et al., 2017). The final 785
average resolution is 2.7 Å for the Nsp1-40S ribosome complex and 3.3 Å for the Nsp1-786
40S-CrPV IRES complex (FSC=0.143). Local refinement was carried out for the head 787
domain of the 40S, which significantly increased the quality of the reconstruction for this 788
domain (Figure S3D). 789
Model building and refinement 790
The structure of the rabbit 40S ribosome was extracted from PDB: 4KZX (Lomakin and 791
Steitz, 2013) and 6SGC (Chandrasekaran et al., 2019). The model of Nsp1 C-terminal 792
domain was manually built in COOT (Emsley et al., 2010). The CrPV IRES structure 793
was extracted form PDB:5IT9 and refined (Murray et al., 2016). The structures of Nsp1-794
40S ribosome complex and Nsp1-IRES-40S ribosome complex were refined with 795
phenix.real_space_refine module in PHENIX (Adams et al., 2010). All structural figures 796
were generated using PyMol (Schrodinger, 2015) and Chimera (Pettersen et al., 2004). 797
798
QUANTIFICATION AND STATISTICAL ANALYSIS 799
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Sample size determination 800
Sample size was determined according to the lab's prior work or similar approaches in 801
the field. 802
Replication 803
All experiments were done with at least three biological replicates. Experimental 804
replications were indicated in detail in methods section and in each figure panel's 805
legend. 806
Standard statistical analysis 807
All statistical methods are described in figure legends and/or supplementary Excel 808
tables. The P values and statistical significance were estimated for all analyses. For 809
example, the unpaired, two-sided, T test was used to compare two groups. One-way 810
ANOVA along with multiple comparisons test, was used to compare multiple groups. 811
Multiple-testing correction was done using false discovery rate (FDR) method. Different 812
levels of statistical significance were accessed based on specific p values and type I 813
error cutoffs (0.05, 0.01, 0.001, 0.0001). Data analysis was performed using GraphPad 814
Prism v.8. and/or RStudio. 815
816
List of Supplemental Tables (provided as excel files) 817
Table S1. Oligo sequences used in this study, Related to Figure 1. 818
819
Table S2. Source data and summary statistics of cellular viability effect by 820
introduction of SARS-CoV-2 viral proteins and mutants, Related to Figure1. 821
822
Table S3. Processed Nsp1 mRNA-seq dataset and differential expression 823
analysis, Related to Figure 2. 824
Sup table 3.1 TPM table of Nsp1 mRNA-seq dataset 825
Sup table 3.2 Differential expression Nsp1 vs Vector Control 826
Sup table 3.3 Differential expression Nsp1 Mutant vs Vector Control 827
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Sup table 3.4 Differential expression Nsp1 vs Nsp1 Mutant 828
829
Table S4. DAVID pathway analysis of Nsp1 differentially expressed gene sets, 830
Related to Figure 2. 831
Sup table 4.1 Functional clustering of Nsp1 vs Vector Control highly 832
downregulated genes (q < 1e-30) 833
Sup table 4.2 Functional clustering of Nsp1 vs Nsp1 Mutant highly 834
downregulated genes (q < 1e-30) 835
Sup table 4.3 Functional clustering of Nsp1 vs Vector Control highly upregulated 836
genes (q < 1e-30) 837
Sup table 4.4 Functional clustering of Nsp1 vs Nsp1 Mutant highly upregulated 838
genes (q < 1e-30) 839
Sup table 4.5 Biological processes enrichment of Nsp1 vs Vector Control highly 840
downregulated genes (q < 1e-30) 841
Sup table 4.6 Biological processes enrichment of Nsp1 vs Nsp1 Mutant highly 842
downregulated genes (q < 1e-30) 843
Sup table 4.7 Biological processes enrichment of Nsp1 vs Vector Control highly 844
upregulated genes (q < 1e-30) 845
Sup table 4.8 Biological processes enrichment of Nsp1 vs Nsp1 Mutant highly 846
upregulated genes (q < 1e-30) 847
Sup table 4.9 Gene list of Nsp1 vs Vector Control highly downregulated genes (q 848
< 1e-30) 849
Sup table 4.10 Gene list enrichment of Nsp1 vs Nsp1 Mutant highly 850
downregulated genes (q < 1e-30) 851
Sup table 4.11 Gene list enrichment of Nsp1 vs Vector Control highly upregulated 852
genes (q < 1e-30) 853
Sup table 4.12 Gene list enrichment of Nsp1 vs Nsp1 Mutant highly upregulated 854
genes (q < 1e-30) 855
Sup table 4.13 Gene list of Nsp1 vs Vector Control all downregulated genes (q < 856
0.01) 857
Sup table 4.14 Gene list of Nsp1 vs Vector Control all upregulated genes (q < 858
0.01) 859
860
861
References: 862
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Highlights
ORF screen identified Nsp1 as a major cellular pathogenicity factor of
SARS-CoV-2.
Nsp1 broadly alters the gene expression programs in human cells of lung origin.
Nsp1 inhibits translation by blocking mRNA entry channel on the 40S ribosome.
Nsp1 prevents physiological conformation of the 48S preinitiation complex (PIC).
eTOC Blurb
Yuan et al. used functional and cryo-EM studies to show that SARS-CoV-2 Nsp1
significantly reduces cell viability, induces extensive transcriptome alteration, and
blocks host mRNA access to the ribosome. These results help understand how
Nsp1 suppresses host gene expression and its broad impact as a
comorbidity-inducing factor.
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